 |
PDBsum entry 2vr9
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Cell adhesion
|
PDB id
|
|
|
|
2vr9
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Structural and functional analysis of slit and heparin binding to immunoglobulin-Like domains 1 and 2 of drosophila robo.
|
|
|
Authors
|
|
N.Fukuhara,
J.A.Howitt,
S.A.Hussain,
E.Hohenester.
|
|
|
Ref.
|
|
J Biol Chem, 2008,
283,
16226-16234.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
Abstract
|
|
|
Recognition of the secreted protein Slit by transmembrane receptors of the Robo
family provides important signals in the development of the nervous system and
other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate
(HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling.
Previous studies have shown that the second leucinerich repeat domain of Slit,
D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we
present two crystal structures of Drosophila Robo IG1-2, one of which contains a
bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a
Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114,
Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly
conserved residues in IG2 had no effect on Slit binding. Mutation of conserved
basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2,
reduced binding of Robo IG1-5 to heparin, in full agreement with the
Robo-heparin co-crystal structure. Our collective results, together with a
recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C.,
Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack,
S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ),
reveal a contiguous HS/heparin binding surface extending across the Slit-Robo
interface. Based on the size of this composite binding site, we predict that at
least five HS disaccharide units are required to support Slit-Robo signaling.
|
|
|
|
|
|
|
|
Figure 5.
FIGURE 5. Heparin oligosaccharide bound to Robo IG1-2. A,
cartoon representation of the crystallographically independent
Robo IG1-2 molecules A and B in the monoclinic crystal form. IG1
and IG2 are shown in orange and green, respectively. The bound
heparin oligosaccharide is shown in ball-and-stick
representation, with carbon atoms colored cyan. The C  atoms of
basic residues implicated in heparin binding are indicated by
blue spheres. The side chains of Lys-69 and Arg-117 are defined
by the electron density, whereas those of Lys-121 and Lys-123
are disordered (see text). B, another view of the bound heparin
oligosaccharide, in an orientation roughly perpendicular to that
of panel A. Heparin binding residues are labeled. C, stereo view
of an unbiased simulated annealing omit map contoured at 2.4
 ,
with the final model of the heparin tetrasaccharide superimposed
on the map. D, superposition of the heparin oligosaccharides
observed in different protein-heparin co-crystal structures:
red, Robo IG1-2 (this study); purple, FGF2-FGFR1 complex (33);
green, FGF1-FGFR2 complex (31); pink, hepatocyte growth
factor/scatter factor NK1 fragment (36).
|
|
Figure 6.
FIGURE 6. Composite heparin binding site in the Slit-Robo
complex. Shown is a model of the human Slit2 D2-Robo1 IG1-2
complex, obtained by substituting Robo1 IG1 with crystal form 1
of Robo1 IG1-2 (14). A, surface representation: pale brown,
Robo1 IG1; pale green, Robo1 IG2; pale pink, Slit2 D2. B,
electrostatic surface representation of the same complex (red,
negative potential; blue, positive potential). The positions of
heparin binding residues in Slit D2 (21) and Robo IG1-2 (this
study) are labeled. Human Robo1 residues Lys-81, Arg-131,
Arg-136, and Lys-137 correspond, respectively, to Drosophila
Robo residues Lys-69, Arg-117, Lys-122, and Lys-123. The
location of the bound heparin oligosaccharide in the Robo IG1-2
structure is indicated by a semitransparent circle. A heparin
octasaccharide (dp8) and decasaccharide (dp10) are drawn to
scale and shown in ball-and-stick representation. HS/heparin is
suggested to bind to the extended basic surface across the
Slit-Robo interface (see text).
|
|
|
|
|
|
The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2008,
283,
16226-16234)
copyright 2008.
|
|
|
|
|
|
|
